The objective of this proposal is to identify potentially causal DNA methylation markers that driver breast cancer development. Aberrant DNA methylation plays an important role in breast cancer initiation and progression via inactivation of tumor suppressor genes and activation of oncogenes. Identifying causal epigenetic markers that drive breast cancer development remains a major challenge. Conventional approaches comparing DNA methylation profiles in tumor and adjacent non-tumorous tissue have shown that changes in DNA methylation contribute to deregulation of gene expression in breast cancer. However, such approaches are unable to distinguish causal DNA methylation alterations, which drive tumorigenesis, from reactive ones that are influenced by the tumor. In addition, important cancer-related methylation changes may have been missed when a suboptimal baseline control (adjacent non-tumorous tissue) was used in previous studies. We hypothesize that an individual's genetic susceptibility and environmental risk factors (e.g. diet, lifestyle) contribut to alterations in causal DNA methylation for breast cancer development, and that such causal changes have already taken place in normal breast tissue before tumor occurrence. To test this hypothesis, normal breast tissue from healthy women is required to prospectively assess the effects of these risk factors on DNA methylation. We propose an integrative genomic and epidemiological approach that combines the regulation of DNA methylation in normal breast tissue and differential DNA methylation profiling in tumor and normal breast tissue. Specifically, we will generate and integrate multiple layers of data on environmental exposures and genome-wide genomic variations (germline and tissue-specific DNA methylation variations). We will examine the impact of genetic and environmental regulation on DNA methylation in normal breast tissue, and determine the association of these epigenetic changes with breast cancer development and occurrence. Normal breast tissue from healthy women will be used as the most desirable baseline control in comparison with tumor, as well as the most desirable target tissue to identify changes in potentially causal DNA methylation markers before tumor occurrence. We are uniquely positioned to conduct the proposed research as we have ready access to unique normal breast tissue through the Susan G. Komen Tissue Bank at Indiana University Simon Cancer Center. This resource represents the only biorepository in the world for large numbers of epidemiologically annotated, optimally prepared normal breast tissue specimens (>3200) from healthy women in a wide range of ages and environmental exposures. This project will contribute significantly to our understanding of the key, early biological processes during breast cancer development, in particular the complex mechanisms among genetic variation, environmental and lifestyle risk factors, and epigenetic changes. It will lead t breakthroughs for breast cancer management in developing novel, more effective prevention and treatment strategies that specifically target causal DNA methylation markers.